The main goal of the proposed studies is to define the molecular signaling pathways whereby activated Ras proteins induce a novel form of non-apoptotic cell death in human glioblastoma cells. Gliomas are notoriously resistant to apoptotic death. Therefore, the long-term goal of this research is to uncover molecular targets that can be manipulated in a therapeutic context to activate non-apoptotic death in this type of cancer. These studies were prompted by the observation that ectopic expression of activated H-Ras or K-Ras in glioblastoma cells causes accumulation of cytoplasmic vacuoles that ultimately disrupt cell viability. The vacuoles are distinct from autophagosomes and may arise from late endosome or lysosome compartments. Active Ras produces similar effects in nine different human glioma cell lines, and the phenotype is recapitulated in stable glioblastoma cell lines where Ras is conditionally expressed. The unusual effects of Ras depend on its membrane association, but are independent from its stimulation of well- known effectors like Raf or PI3K. Preliminary studies indicate that activated Rac1, but not RhoA or Cdc42, can mimic the effects of activated Ras, whereas dominant-negative Rac1 blocks the Ras-induced phenotype. These findings lead to the central hypothesis that Ras activates Rac1-dependent effector pathways in glioblastoma to cause lethal disruptions of endosome or lysosome morphogenesis. To test this hypothesis, studies will focus on four specific aims: (Aim 1) We will define the defective organelles and trafficking events that contribute to non-apoptotic death in human glioblastoma cells. This will include identification of membrane compartments involved in the biogenesis of the vacuoles and delineation of alterations in vesicular trafficking. (Aim 2) We will evaluate the effects of Ras and Rac1 expression on the growth and viability of glioblastoma xenografts in mice, using established cell lines for conditional expression of the proteins. (Aim 3) We will identify the specific Ras effector pathway(s) required for activated Ras to elicit the vacuolar phenotype, focusing on Ras-regulated nucleotide exchange factors (e.g., Tiam1, RasGRF) that activate Rac1 signaling. This will involve the use of dominant-negative mutants, RNAi-mediated gene silencing, and affinity isolation and proteomic characterization of Ras binding partners. (Aim 4) We will define the downstream molecular connections between Rac1 signaling pathway(s) and the endo-lysosomal trafficking machinery in glioblastoma. These studies will focus on Rac1 interactions with Rab GTPases and phosphoinositide 4-phosphate 5'-kinases, which regulate trafficking in early and late endocytic pathways. These studies will contribute to a better understanding of connections between Ras and Rac1 signaling pathways and endo-lysosomal function, and they will provide new knowledge about a novel and poorly understood form of non-apoptotic death with potential clinical significance for treatment of brain tumors.